Four distinct areas were studied in this project in FY2019: 1. The transcription factor Helios is expressed in a large subset of Foxp3+ Tregs. We previously proposed that Helios is a marker of thymic derived Treg (tTreg), while Helios- Treg were induced from Foxp3- T conventional (Tconv) cells in the periphery (pTreg). To compare the two Treg subpopulations, we generated Helios-GFP reporter mice and crossed them to Foxp3-RFP reporter mice. The Helios+ Treg population expressed a more activated phenotype, had a slightly higher suppressive capacity in vitro and expressed a more highly demethylated TSDR, but were equivalent in their ability to suppress inflammatory bowel disease in vivo. However, Helios+ Treg more effectively inhibited the proliferation of activated, autoreactive splenocytes from scurfy mice. When Helios+ and Helios- Treg were transferred to lymphoreplete mice, both populations maintained comparable Foxp3 expression, but Foxp3 expression was less stable in Helios- Treg when transferred to lymphopenic mice. Gene expression profiling demonstrated a large number of differentially expressed genes and showed that Helios- Treg expressed certain genes normally expressed in CD4+Foxp3- T cells. TCR repertoire analysis indicated very little overlap between Helios+ and Helios- Treg. Thus, Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires. 2. Helios expression in Foxp3+ regulatory T (Treg) cells is required for the maintenance of their suppressive phenotype as mice with a selective deficiency of Helios in Treg cells spontaneously develop autoimmunity. However, mice with a deficiency of Helios in all T cells do not exhibit autoimmunity, despite the defect in the suppressor function of their Treg cell population, suggesting that Helios also functions in non-Treg cells. Although Helios is expressed in a small subset of CD4+Foxp3- and CD8+ T cells and its expression is upregulated upon T cell activation, its function in non-Treg cells remains unknown. To examine the function of Helios in CD4+Foxp3- T cells, we transferred Helios-sufficient or -deficient naive CD4+Foxp3-TCR transgenic T cells to normal recipients and examined their capacity to respond to their cognate antigen. Surprisingly, Helios-deficient CD4+ T cells expanded and differentiated into Th1 or Th2 cytokine-producing effectors in a manner similar to wild-type TCR transgenic CD4+ T cells. However, the primed Helios-deficient cells failed to expand upon secondary challenge with Ag. The tolerant state of the Helios-deficient memory T cells was not cell-intrinsic, but was due to a small population of Helios-deficient naive T cells that had differentiated into Ag-specific peripheral Treg cells that suppressed the recall response in an Ag-specific manner. These findings demonstrate that Helios plays a role in the determination of CD4+ T cell fate. 3. Eos (lkzf4) is a member of the Ikaros family of transcription factors and is preferentially expressed in Treg cells. However, the role of Eos in Treg function is controversial. One study using siRNA knockdown of Eos demonstrated that it was critical for Treg suppressor function. In contrast, Treg from mice with global deficiency of Eos had normal Treg function in vitro and in vivo. To further dissect the function of Eos in Tregs, we generated mice with a conditional knockout of Eos in Treg cells (cKO). Deletion of Eos in Treg resulted in activation of CD4+Foxp3- and CD8+ T cells at the age of 3 months, cellular infiltration in non-lymphoid tissues, hyperglobulinemia, and anti-nuclear antibodies. While Tregs from Eos cKO mice displayed normal suppressive function in vitro, Eos cKO mice developed severe Experimental Autoimmune Encephalomyelitis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG) and Eos cKO Treg were unable to suppress Inflammatory Bowel Disease (IBD). Eos cKO mice had decreased growth of the transplantable murine adenocarcinoma MC38 tumor accompanied by enhanced IFN-gamma/TNF-alpha production by CD8+ T cells in tumor draining lymph nodes. Mice with a global deficiency of Eos or a deficiency of Eos only in T cells developed autoimmunity at a much older age (12 months or 7- 8 months, respectively).Taken together, Eos appears to play an essential role in multiple aspects of Treg suppressor function, but also plays an as yet unknown role in the function of CD4+Foxp3- and CD8+ T cells and potentially in non-T cells. 4. While both Treg development in Helios Foxp3 cKO mice and their in vitro suppressor function are normal, the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation with a Th1 phenotype, hypergammaglobulinemia, and enhanced germinal center formation. Initially, we observed significant lymphocytic infiltrates only in the salivary gland and not in any other organs typically affected by Treg dysregulation. Strikingly, the mice developed lipodystrophy, hepatic steatosis and insulin resistance. Further analysis revealed a significant lymphocytic infiltrate in both the inguinal and perigonadal adipose tissue, indicating autoimmune mediated destruction of the white adipose tissue (WAT). We have further shown that the lymphocytic infiltrate specific to WAT can be transferred from mice with lipodystrophy to immunodeficient mice, confirming the autoimmune nature of the lipodystrophy. Thus, Helios deficiency in Treg disrupts immune homeostasis in the adipose tissue, leading to the destruction of WAT and redirection of lipids to the liver, and ultimately causes metabolic dysfunction. This model represents the first description of an autoimmune lipodystrophy.